Computational analysis and in vitro evaluation of TMF 104, for its antioxidant, antimicrobial, and anticancer efficacies.

Benzylidene chromanones are small molecules, structurally similar to active phytochemicals. Herein, we report one novel benzylidene chromanone, TMF 104, for its bio-efficacies. Its computational docking for Vanin-1, antioxidant, free radical scavenging capacities, antimicrobial effects, and anticancer efficacy were analyzed. TMF 104 predicated strong binging to Vanin-1 protein with a docking energy of -8.1 kcal/mol. The compound dose-dependently exhibited free-radical scavenging and antioxidant activities when tested in vitro. The compound also had remarkable activity against Salmonella typhimurium, Enterococcus faecalis, Staphylococcus aureus, and Escherichia coli with minimum inhibitory concentration values of 1.5, 2.0, 12.5, and 13.5 µg/ml, respectively. The compound was also effective against Bacillus cereus and Pseudomonas aeruginosa albeit at higher concentrations. TMF 104 dose-dependently inhibited the proliferation of MCF-7, NCI H460, and Caki-1 cells with respective GI50 values of 24.51, 21.95, and 32.95 µg/ml, whereas the compound was toxic to normal Vero cells at much higher concentration of 264.70 µg/ml. The compound also aided in apoptosis and increased the sub G0 /G1 phase of the cell cycle in all three cancer cells tested. Our study identified a novel, potent benzylidene analogue with potent antioxidant, antimicrobial, and anticancer activities, which drives further attention for further research.

FNF-12, a novel benzylidene-chromanone derivative, attenuates inflammatory response in in vitro and in vivo asthma models mediated by M2-related Th2 cytokines via MAPK and NF-kB signaling.

BACKGROUND AND AIM: This study evaluates a novel benzylidene-chromanone derivative, FNF-12, for efficacy in in vitro and in vivo asthma models. METHODS: Rat basophilic leukemia (RBL-2H3) and acute monocytic leukemia (THP-1)-derived M2 macrophages were used. Human whole blood-derived neutrophils and basophils were employed. Flow cytometry was used for studying key signalling proteins. Platelet activation factor (PAF)-induced asthma model in guinea pigs was used for in vivo studies. RESULTS: The chemical structure of FNF-12 was confirmed with proton-nuclear mass resonance (NMR) and mass spectroscopy. FNF-12 controlled degranulation in RBL-2H3 cells with an IC50 value of 123.7 nM and inhibited TNF-α release from these cells in a dose-responsive way. The compound effectively controlled the migration and elastase release in activated neutrophils. IC50 value in the FcεRI-basophil activation assay was found to be 205 nM. FNF-12 controlled the release of lipopolysaccharide (LPS)-induced interleukin-10, I-309/CCL1 and MDC/CCL22 in THP-1 derived M2 macrophages. The compound suppressed LPS-induced mitogen activated protein kinase (MAPK)-p-p38 and nuclear factor kappa B(NF-kB)-p-p65 expression in these cells. A dose-dependent decrease in the accumulation of total leucocytes, eosinophils, neutrophils and macrophages was observed in PAF-induced animal models. CONCLUSION: FNF-12 was able to control the inflammatory responses in in vitro and in vivo asthma models, which may be driven by controlling M2-related Th2 cytokines via MAPK and NF-kB signaling.

Structure activity evaluation and computational analysis identify potent, novel 3-benzylidene chroman-4-one analogs with anti-fungal, anti-oxidant, and anti-cancer activities.

SIGNIFICANCE: 3-Benzylidene chroman-4-ones share close homology with naturally occurring bioactive compounds. OBJECTIVES: This study evaluated the antifungal, antioxidant, and anticancer activities of novel 3-benzylidene chromanone analogs with respect to their structure-activity relationships. METHODS: Compounds 45e-64e were synthesized inhouse. Aspergillus niger (MTCC 1344) Aspergillus flavus and Botrytis cinerea were the fungal strains tested. Computational docking analysis was carried out for vanin-1, estrogen receptor (ER), and Akt proteins using Auto-dock vina. Free radical scavenging and total antioxidant capacity was analyzed using spectrophotometric methods. MCF-7 (breast cancer) cell line was used for anticancer assays. Flow cytometry was used to detect cell cycle and apoptosis. RESULTS: Out of the twenty compounds screened, compounds 47e, 50e, 52e, 57e, and 61e that possessed either methoxy and ethoxy/methyl/isopropyl group exhibited very good activity against all fungi. Compounds possessing methoxy group alone showed moderate activity and compounds devoid of methoxy, and ethoxy groups did not show any activity. When computationally analyzed against target proteins for antioxidant properties, the compounds exhibited excellent binging efficacy to vanin-1 and ERs. These predictions were translated in the in vitro free-radical scavenging and antioxidant assays. The compounds exhibited anti-proliferative efficacy in breast cancer cell line, increased the sub-G0/G1 cell cycle populations and total apoptosis in MCF-7 cells. Additionally, the compounds also depicted excelling binging energy when computationally analyzed for Akt enzyme binding. CONCLUSION: In summary, our study identified potential analogs of 3-benzylidene chroman-4-one molecules with excellent anti-fungal, anti-oxidant, and anticancer activities which demand further research for drug developments.

Anticancer efficacy of 3-(4-isopropyl) benzylidene-8-ethoxy, 6-methyl, chroman-4-one (SBL-060), a novel, dual, estrogen receptor-Akt kinase inhibitor in acute myeloid leukemia cells.

Estrogen receptor (ER) α is expressed in a subset of patient-derived acute myeloid leukemia (AML) cells, whereas Akt is predominantly expressed in most types of AML. Targeting AML with dual inhibitors is a novel approach to combat the disease. Herein, we examined a novel small molecule, 3-(4-isopropyl) benzylidene-8-ethoxy,6-methyl, chroman-4-one (SBL-060), capable of targeting AML cells by inhibiting ERα and Akt kinase. The chemical properties of SBL-060 were identified by proton nuclear magnetic resonance (1H-NMR), 13C-NMR, and mass spectroscopy. In silico docking was performed using an automated protocol with AutoDock-VINA. THP-1 and HL-60 cell lines were differentiated using phorbol 12-myristate 13-acetate. ERα inhibition was assessed using ELISA. The MTT assay assessed cell viability. Flow cytometry was performed for cell cycle, apoptosis, and p-Akt analyses. Chemical analysis identified the compound as 3-(4-isopropyl) benzylidene-8-ethoxy,6-methyl, chroman-4-one, which showed high binding efficacy toward ER, with a ΔGbinding score of -7.4 kcal/mol. SBL-060 inhibited ERα, exhibiting IC50 values of 448 and 374.3 nM in THP-1 and HL-60 cells, respectively. Regarding inhibited cell proliferation, GI50 values of SBL-060 were 244.1 and 189.9 nM for THP-1 and HL-60 cells, respectively. In addition, a dose-dependent increase in sub G0/G1 phase cell cycle arrest and total apoptosis was observed after treatment with SBL-060 in both cell types. SBL-060 also dose-dependently increased the p-Akt-positive populations in both THP-1 and HL-60 cells. Our results indicate that SBL-060 has excellent efficacy against differentiated AML cell types by inhibiting ER and Akt kinase, warranting further preclinical evaluations.

Computational and in vitro characterization of ICY-5: A potential candidate promoting mitochondrial apoptosis via the c-MET and STAT3 pathways.

Targeted chemotherapy remains the primary choice in controlling various forms of breast cancer (BC) due to its heterogenous gene expressions in various subtypes. In silico and in vitro evaluation of ICY-5, a novel arylidene analogue against c-MET, was performed. ICY-5 exhibited a docking score of -9.6 kcal/mol in inactive conformation and, - 8.6 kcal/mol in active conformation for c-MET. ICY-5 inhibited c-MET enzyme with an IC50 of 34.34 nM. The compound effectively inhibited MDA-MB 231 and MCF-7 cell proliferation, with GI50 values of 62.61 and 75.31 nM, respectively, and hepatocyte growth factor (HGF)/R c-MET phosphorylation with IC50 s of 71.41 and 83.77 nM, respectively. ICY-5 dose-dependently inhibited HGF-induced transmigration, cell scattering, invasion and altered cell cycle. An increase in apoptotic populations of these cells, with a dose-dependent decease in phosphorylation of STAT3 protein was observed. Furthermore, ICY-5 upregulated the caspase-3, caspase-9, Bcl-2-associated X and survivin, and downregulated Bcl-2, vascular endothelial growth factor, matrix metalloproteinase-2 (MMP-2), and MMP-9 in both BC cell lines. In summary, ICY-5 exhibited excellent efficacy in BC cells, targeting c-MET/SAT-3-mediated mitochondrial apoptosis. Further research will be required to ascertain ICY-5 suitability as a targeted chemotherapeutic against multiple forms of BC.

Biallelic loss-of-function variants in TBC1D2B cause a neurodevelopmental disorder with seizures and gingival overgrowth.

The family of Tre2-Bub2-Cdc16 (TBC)-domain containing GTPase activating proteins (RABGAPs) is not only known as key regulatorof RAB GTPase activity but also has GAP-independent functions. Rab GTPases are implicated in membrane trafficking pathways, such as vesicular trafficking. We report biallelic loss-of-function variants in TBC1D2B, encoding a member of the TBC/RABGAP family with yet unknown function, as the underlying cause of cognitive impairment, seizures, and/or gingival overgrowth in three individuals from unrelated families. TBC1D2B messenger RNA amount was drastically reduced, and the protein was absent in fibroblasts of two patients. In immunofluorescence analysis, ectopically expressed TBC1D2B colocalized with vesicles positive for RAB5, a small GTPase orchestrating early endocytic vesicle trafficking. In two independent TBC1D2B CRISPR/Cas9 knockout HeLa cell lines that serve as cellular model of TBC1D2B deficiency, epidermal growth factor internalization was significantly reduced compared with the parental HeLa cell line suggesting a role of TBC1D2B in early endocytosis. Serum deprivation of TBC1D2B-deficient HeLa cell lines caused a decrease in cell viability and an increase in apoptosis. Our data reveal that loss of TBC1D2B causes a neurodevelopmental disorder with gingival overgrowth, possibly by deficits in vesicle trafficking and/or cell survival.

Efficacy of oncolytic mutants targeting pRb and p53 pathways is synergistically enhanced when combined with cytotoxic drugs in prostate cancer cells and tumor xenografts.

Replication-selective oncolytic adenoviruses have proven safety records with promising clinical outcomes. However, strategies to improve efficacy are still required. Here we report greatly improved antitumor efficacy for both attenuated (dl1520) and highly potent (dl922­947) oncolytic mutants in combination with the current standard of care for late-stage hormone-independent prostate cancers, mitoxantrone or docetaxel. In agreement with previous reports, dl922­947 had superior potency compared with dl1520 both as a single agent and in combination with cytotoxic drugs. The dl922­947 mutant caused significant synergistic cell killing in both drug-insensitive and -sensitive prostate cancer cell lines, PC3 and DU145, respectively, when combined with docetaxel or mitoxantrone. The magnitude of the synergistic response was greatest for dl1520 whereas overall efficacy was greatest for dl922­947, and the latter was also more efficacious in vivo in prostate cancer models. In DU145 and PC3 cells increased viral uptake (up to 9- and 8-fold, respectively), E1A expression, and altered cell cycle progression contributed to the synergistic cell killing. A similar trend was also detected in LNCaP cells. Potent E1A expression was essential for the response. In murine xenograft models (DU145 and PC3) tumor growth inhibition was improved when suboptimal doses of docetaxel and viral mutants were combined. These findings demonstrate that the efficacy of highly potent oncolytic mutants such as dl922­947 that target the retinoblastoma protein (pRb) pathway could be further enhanced even with low drug doses, and support the deletion of the E1ACR2 region in future candidate adenoviruses for treatment of hormone-independent prostate cancers.

Indirect recruitment of a CD40 signaling pathway in dendritic cells by B7-DC cross-linking antibody modulates T cell functions.

The human IgM B7-DC XAb protects mice from tumors in both therapeutic and prophylactic settings. Its mechanism of action is mediated by its binding to B7-DC/PD-L2 molecules on the surface of dendritic cells (DCs) to induce a multimolecular cap and subsequent activation of signaling cascades that determine a unique combination of DC phenotypes. One such phenotype, the B7-DC XAb-induced antigen accumulation in mTLR-matured DCs, has been linked to signaling through TREM-2, but the signals required for other DC phenotypes critical for the therapeutic effects in animal models remain unclear. Here, FRET and co-immunoprecipitation studies show that CD40 is recruited to the multi-molecular complex by B7-DC XAb. Signals emanating from CD40 are important, as CD40(-/-) DCs treated with B7-DC XAb (DC(XAb)) activated DAP12, but failed to activate NFkappaB, and were not protected from cell death upon cytokine withdrawal or treatment with Vitamin D(3). CD40(-/-) DC(XAb) also failed to secrete IL-6 and were unable to support the conversion of T regulatory cells into IL-17+ effector T cells in vitro. Importantly, the expression of CD40 was required for the overall ability of B7-DC XAb to induce anti-tumor CTL, to provide protection from a number of tumor types, and for DC(XAb) to be effective anti-tumor vaccines in vivo. These results indicate that B7-DC XAb modulation of DC phenotypes is through its ability to indirectly recruit common signaling molecules and elements of their endogenous signaling pathways through targeted binding to a cell-specific surface determinant.

TREM-2 mediated signaling induces antigen uptake and retention in mature myeloid dendritic cells.

Myeloid dendritic cells (mDC) activated with a B7-DC-specific cross-linking IgM Ab (B7-DC XAb) take up and retain Ag and interact with T cell compartments to affect a number of biologic changes that together cause strong antitumor responses and blockade of inflammatory airway disease in animal models. The molecular events mediating the initial responses in mDC remain unclear. In this study we show that B7-DC XAb caused rapid phosphorylation of the adaptor protein DAP12 and intracellular kinases Syk and phospholipase C-gamma1. Pretreatment of mDC with the Syk inhibitor piceatannol blocked B7-DC XAb-induced Ag uptake with a concomitant loss of tumor protection in mice. Vaccination with tumor lysate-pulsed wild-type B7-DC XAb-activated mDC, but not TREM-2 knockout XAb-activated mDC, protected mice from lethal melanoma challenge. Multimolecular caps appeared within minutes of B7-DC XAb binding to either human or mouse mDC, and FRET analysis showed that class II, CD80, CD86, and TREM-2 are recruited in tight association on the cell surface. When TREM-2 expression was reduced in wild-type mDC using short hairpin RNA or by using mDC from TREM-2 knockout mice, in vitro DC failed to take up Ag after B7-DC XAb stimulation. These results directly link TREM-2 signaling with one change in the mDC phenotype that occurs in response to this unique Ab. The parallel signaling events observed in both human and mouse mDC support the hypothesis that B7-DC cross-linking may be useful as a therapeutic immune modulator in human patients.

Reprogrammed FoxP3+ T regulatory cells become IL-17+ antigen-specific autoimmune effectors in vitro and in vivo.

Lymphocyte differentiation from naive CD4(+) T cells into mature Th1, Th2, Th17, or T regulatory cell (Treg) phenotypes has been considered end stage in character. In this study, we demonstrate that dendritic cells (DCs) activated with a novel immune modulator B7-DC XAb (DC(XAb)) can reprogram Tregs into T effector cells. Down-regulation of FoxP3 expression after either in vitro or in vivo Treg-DC(XAb) interaction is Ag-specific, IL-6-dependent, and results in the functional reprogramming of the mature T cell phenotype. The reprogrammed Tregs cease to express IL-10 and TGFbeta, fail to suppress T cell responses, and gain the ability to produce IFN-gamma, IL-17, and TNF-alpha. The ability of IL-6(+) DC(XAb) and the inability of IL-6(-/-) DC(XAb) vaccines to protect animals from lethal melanoma suggest that exogenously modulated DC can reprogram host Tregs. In support of this hypothesis and as a test for Ag specificity, transfer of DC(XAb) into RIP-OVA mice causes a break in immune tolerance, inducing diabetes. Conversely, adoptive transfer of reprogrammed Tregs but not similarly treated CD25(-) T cells into naive RIP-OVA mice is also sufficient to cause autoimmune diabetes. Yet, treatment of normal mice with B7-DC XAb fails to elicit generalized autoimmunity. The finding that mature Tregs can be reprogrammed into competent effector cells provides new insights into the plasticity of T cell lineage, underscores the importance of DC-T cell interaction in balancing immunity with tolerance, points to Tregs as a reservoir of autoimmune effectors, and defines a new approach for breaking tolerance to self Ags as a strategy for cancer immunotherapy.

Fast-tracked CTL: rapid induction of potent anti-tumor killer T cells in situ.

Current strategies to elicit cytolytic T cell responses specific for tumor-associated or over-expressed self antigens rely on multiple immunizations and in vitro expansion schemes. Here we report the in vivo induction of activated tumor-specific CD8(+) CTL just 6 days after treatment with the IgM immune modulator B7-DC XAb. Antibody treatment of mice at the time of tumor challenge elicited potent CTL with a specificity that distinguished between MHC-compatible tumors. Remarkably, these effector cells were not generated by the extensive proliferation of naive CTL precursors, though their induction required CD4(+) T cell help and classical B7 costimulatory signals. Tumor targets were recognized and lysed in an MHC-restricted, perforin-dependent manner, indicating that these rapidly induced effectors resemble traditionally defined CTL, despite the finding that strong increases in the expression of the effector/memory marker CD44 and the activation marker CD69 were not elicited. These CTL were induced in animals bearing well-established tumors and resulted in anti-tumor protection, underscoring the therapeutic potential of this type of effector T cell population in cancer patients.

Induction of a Th1 response from Th2-polarized T cells by activated dendritic cells: dependence on TCR:peptide-MHC interaction, ICAM-1, IL-12, and IFN-gamma.

Dendritic cells (DC) are important regulators of T cell immunity. The degree of stimulation, the pattern of costimulatory molecules expressed, and the cytokines secreted by DC dictate the nature of the effector and memory cells generated, particularly with respect to their Th1 or Th2 phenotypes. In this study, we demonstrate that the addition of activated DC to spleen cultures containing established Th2-polarized CD4(+) T cells was sufficient to suppress Th2 and induce Th1 cytokines in a recall response, a phenomenon referred to as phenotype reversal. The ability of activated DC to induce phenotype reversal displayed exquisite Ag specificity. The DC activator B7-DC cross-linking Ab (XAb) was >10,000-fold more efficient at inducing phenotype reversal than the TLR agonists CpG-oligodeoxynucleotide and Gardiquimod. Characterization of the mechanisms governing phenotype reversal revealed the requirement for cognate interaction between the TCR:peptide-MHC complex, the expression of the costimulation/adhesion molecule ICAM-1, and secretion of IL-12 and IFN-gamma by the activated DC. The requirement for the costimulation/adhesion molecule SLAM (signaling lymphocytic activation molecule) was found to be quantitative. Thus, activation of DC, particularly by crosslinking B7-DC, can modulate well-established Th2 T cell responses in an Ag-specific manner. Because the regulation of mouse and human DC by B7-DC XAb overlaps in several significant ways, immune modulation with B7-DC XAb is a potential strategy for treating Th2-mediated diseases.

B7-DC/PD-L2 cross-linking induces NF-kappaB-dependent protection of dendritic cells from cell death.

Cross-linking cell surface molecules with IgM Abs is a specific approach for activating cells in vitro or in vivo. Dendritic cells (DC) activated with a human B7-DC (PD-L2)-specific IgM Ab can induce strong antitumor responses and block inflammatory airway disease in experimental models, yet the Ab-mediated molecular events promoting these responses remain unclear. Analysis of human or mouse DC treated with the B7-DC cross-linking Ab revealed PI3K-dependent phosphorylation of AKT accompanied by mobilization of NF-kappaB. Ab-activated DC up-regulated expression of cytokine and chemokine genes in an NF-kappaB-dependent manner. Importantly, PI3K-->AKT-->NF-kappaB activation was found to be indispensable for B7-DC cross-linking Ab-mediated protection of DC from cell death caused by cytokine withdrawal. Although other DC activators similarly protect DC from cell death, a synergy between cross-linking B7-DC and ligating RANK was observed. The parallel signaling events induced in human and mouse DC demonstrate that activation of cells using IgM Ab results in a response governed by a common mechanism and support the hypothesis that B7-DC cross-linking using this Ab may provide beneficial therapeutic immune modulation in human patients similar to those seen in animal models.

Induction of a gene expression program in dendritic cells with a cross-linking IgM antibody to the co-stimulatory molecule B7-DC.

While bivalent antibodies can block ligand-receptor interactions, IgM pentamers efficiently cross-link cell surface targets and evoke physiological responses. We have described one such interaction between an IgM antibody (Ab) and the B7-DC costimulatory molecule expressed by dendritic cells that induces strong antitumor immunity and modulates pathogenic responses associated with allergic asthma. Progressive changes in gene expression in dendritic cells activated by an IgM B7-DC cross-linking Ab resulted in the increased expression in 350 genes and decreased expression of more than 200 genes over the course of 24 h following Ab treatment. In particular, up-regulation of the caspase inhibitor FLIP and the chemokine receptor CCR7, and the down-regulation of the CXCR4 receptor provide a mechanistic basis of Ab-induced survival and enhanced migration into draining lymph nodes. Increased expression of both cell surface and secreted molecules known to be mediators of the immunomodulatory properties of dendritic cells was detected at both the levels of RNA and protein expression. This analysis documents the ability of IgM Ab to activate a gene expression cascade leading to important biological changes in cellular function and provides mechanistic insight into the potent immunomodulatory properties attributed to this Ab.

STAT4- and STAT6-signaling molecules in a murine model of multiple sclerosis.

Epidemiological studies suggest that an environmental factor (possibly a virus) acquired early in life may trigger multiple sclerosis (MS). The virus may remain dormant in the central nervous system but then becomes activated in adulthood. All existing models of MS are characterized by inflammation or demyelination that follows days after virus infection or antigen inoculation. While investigating the role of CD4+ T cell responses following Theiler's virus infection in mice deficient in STAT4 or STAT6, we discovered a model in which virus infection was followed by demyelination after a very prolonged incubation period. STAT4-/- mice were resistant to demyelination for 180 days after infection, but developed severe demyelination after this time point. Inflammatory cells and up-regulation of Class I and Class II MHC antigens characterized these lesions. Virus antigen was partially controlled during the early chronic phase of the infection even though viral RNA levels remained high throughout infection. Demyelination correlated with the appearance of virus antigen expression. Bone marrow reconstitution experiments indicated that the mechanism of the late onset demyelination was the result of the STAT4-/- immune system. Thus, virus infection of STAT4-/- mice results in a model that may allow for dissection of the immune events predisposing to late-onset demyelination in MS.

Urethral catheter induced pseudoaneurysm of the bulbar artery.

Injuries of the urethra due to urethral catheter have been well recognised. We report a case of pseudoaneurysm of the artery to the bulb of the penis which formed due to necrosis of the arterial wall secondary to prolonged pressure due to a wrongly placed urethral catheter. The catheter was inflated in the bulbar urethra and left during surgery for 4 hours. The patient developed intermittent severe urethral bleeding. An angiogram of internal iliac artery showed a pseudo aneurysm involving the bulbar artery with arteriovenous communication. Super selective embolization of the feeding vessel was performed with cessation of the blood fl ow immediately. To our knowledge, pseudoaneurysm of the bulbar artery of penis has not previously been described.

Immunotherapeutic potential of B7-DC (PD-L2) cross-linking antibody in conferring antitumor immunity.

A naturally occurring human antibody potentiates dendritic cell function on cross-linking B7-DC (PD-L2), supporting robust T-cell responses in vitro. Moreover, treatment of dendritic cells with B7-DC cross-linking antibody resulted in secretion of interleukin-12, suggesting a TH1 polarization of this response. Here we show an in vivo immunotherapeutic effect of this B7-DC cross-linking antibody using a poorly immunogenic B16 melanoma tumor model. Treatment of mice systemically with antibody at the time of tumor cell engraftment prevented tumor growth in a CD4 and CD8 T-cell-dependent manner. The protective effect of B7-DC cross-linking antibody treatment was independent of endogenous antibody responses. Tumor-specific CTL precursors could be isolated from lymph nodes draining the tumor site in animals treated with B7-DC cross-linking antibody, but not from those treated with isotype control antibodies. The elicited antitumor responses in vivo were specific and long-lasting. More strikingly, treatment of mice with B7-DC cross-linking antibody after the tumors were established in the lungs resulted in protection in a CD8-, perforin-, and granzyme B-dependent fashion. Depletion of natural killer cells did not block the effects of treatment with B7-DC cross-linking antibody. Together, these findings demonstrate that cross-linking B7-DC with the human IgM antibody sHIgM12 can induce a protective immune response against a weakly antigenic experimental tumor and therefore has potential as a novel immunotherapeutic approach for treating cancer.